Intranasal administration of mesenchymal stem cells ameliorates the abnormal dopamine transmission system and inflammatory reaction in the R6/2 mouse model of Huntington disease

Intrastriatal administration of mesenchymal stem cells (MSCs) has shown beneficial effects in rodent models of Huntington disease (HD). However, the invasive nature of surgical procedure and its potential to trigger the host immune response may limit its clinical use. Hence, we sought to evaluate the non-invasive intranasal administration (INA) of MSC delivery as an effective alternative route in HD. GFP-expressing MSCs derived from bone marrow were intranasally administered to 4-week-old R6/2 HD transgenic mice. MSCs were detected in the olfactory bulb, midbrain and striatum five days post-delivery. Compared to phosphate-buffered saline (PBS)-treated littermates, MSC-treated R6/2 mice showed an increased survival rate and attenuated circadian activity disruption assessed by locomotor activity. MSCs increased the protein expression of DARPP-32 and tyrosine hydroxylase (TH) and downregulated gene expression of inflammatory modulators in the brain 7.5 weeks after INA. While vehicle treated R6/2 mice displayed decreased Iba1 expression and altered microglial morphology in comparison to the wild type littermates, MSCs restored both, Iba1 level and the thickness of microglial processes in the striatum of R6/2 mice. Our results demonstrate significantly ameliorated phenotypes of R6/2 mice after MSCs administration via INA, suggesting this method as an effective delivering route of cells to the brain for HD therapy

Proposed pathway of kalirin-7-mediated synphilin-1 aggresome formation.

<p>(A) Under normal conditions, misfolded synphilin-1 is mainly accumulated in cytoplasmic small aggregates. (B) When kalirin-7 is overexpressed, it facilitates the recruitment of HDAC6 and the dynein motor complex and acts on microtubule dynamics by stimulating the deacetylase activity of HDAC6, thereby increasing the transportation of synphilin-1 into aggresomes.</p

Characterization of synphilin-1-containing aggregates as aggresomes.

<p>HEK293 cells coexpressing HcRed-synphilin-1 and FLAG-kalirin-7 were fixed 48 h post-transfection and subsequently stained with the indicated antibodies. Arrows indicate the colocalization between synphilin-1 inclusions and γ-tubulin, ubiquitin and Hsp27 while the intermediate filament protein vimentin forms a cage surrounding a pericentriolar core of aggregates. Merged images are shown to the right. <i>Blue</i>, DAPI. <i>Scale bar</i>, 10 µm.</p

Kalirin-7-mediated recruitment of synphilin-1 inclusions into aggresome is blocked by the HDAC inhibitor trichostatin A.

<p>(A) HEK293 cells expressing HcRed-synphilin-1 (a,b,c) or co-expressing HcRed-synphilin-1 and FLAG-kalirin-7 (d,e,f) were incubated in the presence of DMSO (a,d), 1 µM TSA (b,e) or 5 mM NaBu (c,f) for 18 h before being fixed and immunostained with anti-FLAG antibodies. The arrow indicates synphilin-1 cytoplasmic small aggregates. <i>Blue</i>, DAPI. <i>Scale bar</i>, 10 µm. (B) Quantification shows that treatment with the HDAC6 inhibitor TSA counteracts the recruitment of synphilin-1 into aggresomes mediated by kalirin-7, whereas the broad deacetylase inhibitor NaBu does not exert such an effect. The asterisks indicate statistical significance (**<i>P</i>≤0.005). Error bars, S.E.</p

Reduced Motivation in the BACHD Rat Model of Huntington Disease Is Dependent on the Choice of Food Deprivation Strategy

<div><p>Huntington disease (HD) is an inherited neurodegenerative disease characterized by motor, cognitive, psychiatric and metabolic symptoms. Animal models of HD show phenotypes that can be divided into similar categories, with the metabolic phenotype of certain models being characterized by obesity. Although interesting in terms of modeling metabolic symptoms of HD, the obesity phenotype can be problematic as it might confound the results of certain behavioral tests. This concerns the assessment of cognitive function in particular, as tests for such phenotypes are often based on food depriving the animals and having them perform tasks for food rewards. The BACHD rat is a recently established animal model of HD, and in order to ensure that behavioral characterization of these rats is done in a reliable way, a basic understanding of their physiology is needed. Here, we show that BACHD rats are obese and suffer from discrete developmental deficits. When assessing the motivation to lever push for a food reward, BACHD rats were found to be less motivated than wild type rats, although this phenotype was dependent on the food deprivation strategy. Specifically, the phenotype was present when rats of both genotypes were deprived to 85% of their respective free-feeding body weight, but not when deprivation levels were adjusted in order to match the rats' apparent hunger levels. The study emphasizes the importance of considering metabolic abnormalities as a confounding factor when performing behavioral characterization of HD animal models.</p></div

Head entry behavior during delays of the delayed alternation protocol.

<p>The graphs show several aspects of head entries made into the pellet trough during delay steps of the delayed alternation test. Curves were created based on the overall performance at all test ages, as no significant change with age was found for the parameters. (C) concerns the 20-second delay step. Graphs indicate group mean plus standard error. Results from two-way repeated measures ANOVA are shown inside the graphs. Results from <i>post-hoc</i> analysis are indicated in case significant genotype differences were found. * (<i>P</i> < 0.05) ** (<i>P</i> < 0.01) *** (<i>P</i> < 0.001).</p

Kalirin-7 interacts with synphilin-1 <i>in vitro</i> and <i>in vivo</i>.

<p>(A) Mapping of the interacting domain in the kalirin-7 protein. FLAG-kalirin-7 constructs as shown in the diagram were co-transfected with V5-synphilin-1 in HEK293 cells. 24 h after transfection, cells were subjected to immunoprecipitation with anti-FLAG agarose beads and subsequently kalirin-7 and synphilin-1 immunoreactivities were monitored applying anti-FLAG- or anti-V5 antibodies, respectively. IP indicates antibodies used for pulling down target proteins. IB indicates antibodies used for detection in western blot. The figure shows that kalirin-7 co-immunopreciptates with synphilin-1 and that spectrin repeats III and IV of the kalirin-7 protein are crucial for the interaction. Quantification of kalirin-7 fragment expression is shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051999#pone.0051999.s003" target="_blank">Figure S3</a>. (B) Mapping of the binding region in synphilin-1. The indicated V5-synphilin-1 constructs were co-transfected with FLAG-kalirin-7. Synphilin-1 fragments were precipitated with anti-V5 antibodies. The precipitates were then probed with anti-FLAG antibodies to detect co-precipitated kalirin-7. The deletion mapping revealed that amino acids 1–348 of the synphilin-1 protein are crucial for the binding of kalirin-7. The asterisks indicate specific input signals of synphilin-1 fragments. For quantification of synphilin-1 fragment expression please refer to <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051999#pone.0051999.s003" target="_blank">Figure S3</a>. (C) Endogenous synphilin-1 interacts with kalirin-7. Synphilin-1 was precipitated from whole-brain tissues (500 µg) of a wild type mouse with an anti-synphilin-1 antibody (Sigma). The precipitates were probed with a kalirin-7-specific antibody (KALRN from Abcam). Cell lysate of HEK293 cells overexpressed with FLAG-kalirin-7 and V5-synphilin-1 served as positive control. As a negative control brain lysate was subjected to immunoprecipitation without antibody. (D) Overlapping localization of kalirin-7 and synphilin-1 in cell culture. HEK293 cells were transiently transfected with both constructs for 6 h and stained with anti-FLAG and anti-V5 antibodies. The counterstaining was done with YoPro dye. The confocal sections demonstrate that both proteins display a punctate staining in the cytoplasm. Sph1, synphilin-1; Kal7, kalirin-7. <i>Scale bar</i>, 10 µm.</p

Kalirin-7 mediates perinuclear synphilin-1 inclusion formation in a microtubule-dependent manner.

<p>(A) HEK293 cells were cotransfected with HcRed-synphilin-1 and FLAG-kalirin-7. After 36 h cells were incubated with DMSO, 5 µM nocodazole or 10 µM colchicine for 12 h before being subjected to immunofluorescence with anti-FLAG and anti- β-tubulin antibodies. Cells expressing HcRed-synphilin-1 alone served as controls (arrowhead). In cells treated with nocodazole or colchicine, more cytoplasmic small aggregates (arrows) were formed. (B) Quantification (n >250 cells per group) shows that nocodazole and colchicine inhibited the kalirin-7-mediated formation of synphilin-1-containing perinuclear inclusions. P, perinuclear aggregates; C, cytoplasmic small aggregates. The asterisks indicate statistical significance (^**<i>P</i>≤0.005). <i>Error bars</i>, S.E.</p

An HDAC6 deacetylase-dead mutant opposes the formation of synphilin-1 containing aggresomes mediated by kalirin-7.

<p>(A) HEK293 cells were triple-transfected with HcRed-synphilin-1, EGFP-kalirin-7 and FLAG-WT HDAC6 or FLAG-H216A/H611A mutant HDAC6. After 48 h cells were fixed and stained with anti-FLAG antibodies. Only cells co-expressing mutant HDAC6 form more cytoplasmic small aggregates (arrows). (B) Quantification shows that mutant HDAC6 inhibited the kalirin-7-mediated perinuclear synphilin-1 inclusion formation. The asterisks indicate statistical significance (^***<i>P</i>≤0.001). <i>Error bars</i>, S.E.</p

Head entry behavior during delays on the delayed non-matching to position protocol.

<p>The graphs show several aspects of head entries made into the pellet trough during delay steps of the delayed non-matching to position test. Curves were created based on the overall performance on all test ages, as no consistent change with age was found for the parameters. (C) concerns the 25-second delay step. Graphs indicate group mean plus standard error. Results from two-way repeated measures ANOVA are shown inside the graphs. Results from <i>post-hoc</i> analysis are indicated in case significant genotype differences were found. * (<i>P</i> < 0.05) ** (<i>P</i> < 0.01) *** (<i>P</i> < 0.001).</p